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Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector

Gene Therapy volume 17, pages 1244–1252 (2010)Cite this article

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Abstract

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34+ cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

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References

  1. Bagby GC, Alter BP . Fanconi anemia (Review). Semin Hematol 2006; 43: 147–156.

    Article  CAS  PubMed  Google Scholar 

  2. Taniguchi T, D'Andrea AD . Molecular pathogenesis of Fanconi anemia: recent progress (Review). Blood 2006; 107: 4223–4233.

    Article  CAS  PubMed  Google Scholar 

  3. Wang W . Emergence of a DNA-damage response network consisting of Fanconi anaemia and BRCA proteins (Review). Nat Rev Genet 2007; 8: 735–748.

    Article  CAS  PubMed  Google Scholar 

  4. Liu JM, Kim S, Read EJ, Futaki M, Dokal I, Carter CS et al. Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC). Hum Gene Ther 1999; 10: 2337–2346.

    Article  CAS  PubMed  Google Scholar 

  5. Yamada K, Olsen JC, Patel M, Rao KW, Walsh CE . Functional correction of fanconi anemia group C hematopoietic cells by the use of a novel lentiviral vector. Mol Ther 2001; 3: 485–490.

    Article  CAS  PubMed  Google Scholar 

  6. Yamada K, Ramezani A, Hawley RG, Ebell W, Arwert F, Arnold LW et al. Phenotype correction of Fanconi anemia group A hematopoietic stem cells using lentiviral vector. Mol Ther 2003; 8: 600–610.

    Article  CAS  PubMed  Google Scholar 

  7. Cohen-Haguenauer O, Peault B, Bauche C, Daniel MT, Casal I, Levy V et al. In vivo repopulation ability of genetically corrected bone marrow cells from Fanconi anemia patients. Proc Natl Acad Sci USA 2006; 103: 2340–2345.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Kelly PF, Radtke S, von Kalle C, Balcik B, Bohn K, Mueller R et al. Stem cell collection and gene transfer in Fanconi anemia. Mol Ther 2007; 15: 211–219.

    Article  CAS  PubMed  Google Scholar 

  9. Müller LU, Milsom MD, Kim MO, Schambach A, Schuesler T, Williams DA . Rapid lentiviral transduction preserves the engraftment potential of Fanca(−/−) hematopoietic stem cells. Mol Ther 2008; 16: 1154–1160.

    Article  PubMed  Google Scholar 

  10. Jacome A, Navarro S, Rio P, Yañez RM, González-Murillo A, Lozano ML et al. Lentiviral-mediated genetic correction of hematopoietic and mesenchymal progenitor cells from Fanconi anemia patients. Mol Ther 2009; 17: 1083–1092.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Butturini A, Gale RP, Verlander PC, Aldler-Bricner B, Gillio AP, Auerbach AD . Hematologic abnormalities in Fanconi anemia: An International Fanconi Anemia Registry Study. Blood 1994; 84: 1650–1655.

    CAS  PubMed  Google Scholar 

  12. Laufs S, Guenechea G, Gonzalez-Murillo A, Zsuzsanna Nagy K, Luz Lozano M, del Val C et al. Lentiviralvector integration sites in human NOD/SCID repopulating cells. J Gene Med 2006; 8: 1197–1207.

    Article  CAS  PubMed  Google Scholar 

  13. Beard BC, Keyser KA, Trobridge GD, Peterson LJ, Miller DG, Jacobs M et al. Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, and foamy virus. Hum Gene Ther 2007; 18: 423–434.

    Article  CAS  PubMed  Google Scholar 

  14. Alter BP, Knobloch ME, Weinberg RS . Erythropoiesis in Fanconi's anemia. Blood 1991; 78: 602–608.

    CAS  PubMed  Google Scholar 

  15. Waisfisz Q, Morgan NV, Savino M, de Winter JP, van Berkel CG, Hoatlin ME et al. Spontaneous functional correction of homozygous fanconi anaemia alleles reveals novel mechanistic basis for reverse mosaicism. Nat Genet 1999; 22: 379–383.

    Article  CAS  PubMed  Google Scholar 

  16. Gregory Jr JJ, Wagner JE, Verlander PC, Levran O, Batish SD, Eide CR et al. Somatic mosaicism in Fanconi anemia: evidence of genotypic reversion in lymphohematopoietic stem cells. Proc Natl Acad Sci USA 2001; 98: 2532–2537.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Zufferey R, Donello JE, Trono D, Hope TJ . Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 1999; 73: 2886–2892.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Kingsman SM, Mitrophanous K, Olsen JC . Potential oncogene activity of the woodchuck hepatitis post-transcriptional regulatory element (WPRE). Gene Therapy 2005; 12: 3–4.

    Article  CAS  PubMed  Google Scholar 

  19. Levesque JP, Winkler IG, Hendy J, Williams B, Helwani F, Barbier V et al. Hematopoietic progenitor cell mobilization results in hypoxia with increased hypoxia-inducible transcription factor-1 alpha and vascular endothelial growth factor A in bone marrow. Stem Cells 2007; 25: 1954–1965.

    Article  CAS  PubMed  Google Scholar 

  20. Shima H, Takubo K, Iwasaki H, Yoshihara H, Gomei Y, Hosokawa K et al. Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice. Biochem Biophys Res Commun 2009; 378: 467–472.

    Article  CAS  PubMed  Google Scholar 

  21. Gross M, Hanenberg H, Lobitz S, Friedl R, Herterich S, Dietrich R et al. Reverse mosaicism in Fanconi anemia: natural gene therapy via molecular self-correction. Cytogenet Genome Res 2002; 98: 126–135.

    Article  CAS  PubMed  Google Scholar 

  22. Mankad A, Taniguchi T, Cox B, Akkari Y, Rathbun RK, Lucas L et al. Natural gene therapy in monozygotic twins with Fanconi anemia. Blood 2006; 107: 3084–3090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Zhang X, Shang X, Guo F, Murphy K, Kirby M, Kelley P et al. Defective homing is associated with altered Cdc42 activity in cells from patients with Fanconi anemia group A. Blood 2008; 112: 1683–1686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L et al. Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 1998; 72: 9873–9880.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Follenzi A, Ailles LE, Bakovic S, Geuna M, Naldini L . Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet 2000; 25: 217–222.

    Article  CAS  PubMed  Google Scholar 

  26. Yamashita T, Kupfer GM, Naf D, Suliman A, Joenje H, Asano S et al. The fanconi anemia pathway requires FAA phosphorylation and FAA/FAC nuclear accumulation. Proc Natl Acad Sci USA 1998; 95: 13085–13090.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Beard BC, Sud R, Keyser KA, Ironside C, Neff T, Gerull S et al. Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients. Blood 2009; 113: 5094–5103.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by NIH Grants HL085693, DK56465 and DK47754 to HPK. We thank Helen Crawford and Bonnie Larson for help with the preparation of the article. HPK is a Molecular Medicine Investigator and the recipient of the José Carreras/E. Donnall Thomas Endowed Chair for Cancer Research. We received grant support from National Institutes of Health.

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Author notes

  1. P S Becker and J A Taylor: These authors contributed equally to this work.

Authors and Affiliations

  1. Division of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA

    P S Becker, X Zhao & S Chien

  2. Division of Hematology/Oncology, Department of Medicine, Oregon Health Sciences University and Portland VA Medical Center, Portland, OR, USA

    J A Taylor

  3. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    G D Trobridge, B C Beard, J Adair, A Shimamura & H-P Kiem

  4. Division of Medical Oncology, Department of Medicine, University of Washington, Seattle, WA, USA

    G D Trobridge & H-P Kiem

  5. Departments of Microbiology, Immunology and Molecular Genetics and Pediatrics, University of California, Los Angeles, CA, USA

    D B Kohn

  6. Division of Pediatric Hematology/Oncology, Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA

    J E Wagner

  7. Division of Pediatric Hematology/Oncology, Department of Pediatrics, Seattle Children's Hospital, Seattle, WA, USA

    A Shimamura

  8. Department of Pathology, University of Washington, Seattle, WA, USA

    H-P Kiem

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  1. P S Becker
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  2. J A Taylor
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Corresponding author

Correspondence to P S Becker.

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The authors declare no conflict of interest.

Additional information

Portions of this work were presented in preliminary form at the American Society of Gene Therapy and American Society of Hematology meetings, 2008.

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Becker, P., Taylor, J., Trobridge, G. et al. Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector. Gene Ther 17, 1244–1252 (2010). https://doi.org/10.1038/gt.2010.62

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  • DOI: https://doi.org/10.1038/gt.2010.62

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